Project Summary The complement system controls the balance between homeostatic and inflammatory processes. Although abnormal complement activity is strongly associated with macular degenerations, inhibiting this pathway has been unsuccessful in halting vision loss. To date, therapeutic approaches have focused on complete inhibition of extracellular complement activity, with limited insight into how complement proteins modulate retinal health and disease. This is particularly evident in approaches targeting the complement protein C3, the core effector molecule of the complement system, which plays context-dependent roles in the retina. Countering the conventional view of C3 acting solely in the extracellular space, recent studies from our group have identified intracellular C3 activation as a novel mechanism that modulates health of the retinal pigment epithelium (RPE), a primary site of injury in macular degenerations. In stressed or diseased RPE, increased uptake and proteolysis of C3 generates biologically active C3a (?intracellular C3 activation?). C3a in turn activates mTOR, a master regulator of cell health. Chronic mTOR activation is detrimental to cell health because it can reprogram cellular metabolism and cell fate decisions. Based on these exciting studies, we hypothesize that abnormal intracellular complement activation could drive disease pathology by compromising RPE homeostasis. We propose to molecularly dissect the machinery, mechanisms, and consequences of dysregulated intracellular C3a activity in the RPE, and identify potential points of therapeutic intervention to halt this cascade. We will identify the cellular machinery responsible for increased intracellular C3a generation in diseased RPE (Aim 1); investigate the dynamics of C3a signaling via its cognate receptor C3aR (Aim 2); and determine how persistent C3a-C3aR signaling disrupts RPE homeostasis and retinal function (Aim 3). We will use our expertise in innovative high- speed and super-resolution live-cell imaging and mouse models of disease to gain unprecedented spatial and temporal information about intracellular C3 activation and its consequences for retinal health. These studies will aid the development of a unified model that links multiple features of AMD, including cholesterol accumulation, complement activation, metabolic deficits and RPE dedifferentiation. Identifying molecular mechanisms that underlie increased intracellular C3 activation will aid the design of precision therapeutics to safeguard RPE health and retinal function over a lifetime.